Reactive oxygen and nitrogen species, produced enzymatically, are major contributors to mammalian innate immunity. Whereas the cytotoxic mechanisms of reactive oxygen species, such as H2O2 and O2 -, have been extensively studied, the mechanisms by which reactive nitrogen species (e.g. nitric oxide and S-nitrosothiols) kill microbes are relatively unexplored. There is evidence that nitrosative stress induced by reactive nitrogen species is conveyed in significant part by an increase in intracellular S-nitrosylated proteins. This idea is supported by our recent demonstrations of a microbial flavohemoglobin and an evolutionarily conserved Snitrosoglutathione reductase, both of which regulate S-nitrosylation and protect from cell death. The proposed studies are designed to enhance understanding of the molecular basis of nitrosative stress, and in particular protein S-nitrosylation, in the model bacteria E. coli MG1655 and in uropathogenic strains of E. coli. In Aim 1, novel resin-based proteomic methods are employed to determine the specific sites of S-nitrosylation mediated by various nitrosative stimuli induced under physiologically relevant conditions, including an endogenous nitrosative stress that we show is responsible for regulating the S-nitrosylation of bacterial proteins. In Aim 2, we examine the enzymatic mechanisms that regulate nitrosative stress through protein denitrosylation, focusing on a denitrosylase that we have identified in bacteria. In Aim 3, mechanisms of resistance to nitrosative stress are analyzed more broadly by microarray-based profiling of the transcriptional response specific to nitrosative stress, in conjunction with functional and biochemical assays. This analysis will include an evaluation of the role of stress-induced genes in uropathogenic bacteria. In sum, these three Aims converge on the fundamental issue of how nitrosative stress exerts microbicidal activity and how microbes mount a defense. Our studies will provide novel insights into S-nitrosylation-based cellular signaling. They also have direct relevance to human pathophysiology because they are likely to uncover novel pharmacological targets within bacteria and point towards potential strategies to augment innate immunity.